The tumor vaccine therapy is to activate immune system in vivo, particularly killer lymphocytes that play a key role in cellular immune responses, especially cytolytic T lymphocytes (hereinafter abbreviated as “CTL”), to specifically kill tumor cells without damaging normal cells, and to expect prevention of recurrence of the tumor, inhibition of metastasis, or cure of the established tumor.
Various kinds of tumor vaccines have been developed (Pardoll, D. M., Nature Med., 4(5 Suppl), pp. 525-531, 1998). Roughly tumor vaccines can be categorized depending on tumor-specific materials as follows: (1) vaccines wherein a tumor antigenic peptide with a known property is used; (2) vaccines wherein a tumor tissue extract containing an unidentified tumor antigenic peptide is used; (3) vaccines wherein the above peptide is bound to an antigen-presenting cell, especially a dendritic cell with a strong capability of antigen presentation (Nestle, F. O., et al., Nature Med., 4, pp. 328-332, 1998); (4) vaccines wherein a tumor antigenic protein is taken into a dendritic cell and loaded; (5) vaccines wherein a dendritic cell and a tumor cell are fused; (6) vaccines wherein a tumor antigen is bound to a liposome for uptake together with the liposome (Nakanishi, T., et al., Biochem. Biophys. Res. Comm., 240, pp. 793-797, 1997); (7) vaccines wherein a tumor cell, per se, is treated for inactivation with radiation or a fixing agent before administration; (8) vaccines wherein a cytokine gene, having an antigen-presenting cell stimulating effect or a lymphocyte stimulating effect, is introduced into a tumor cell and the cell is administered as a vaccine for a gene therapy, or wherein a tumor antigenic gene is introduced into a suitable cell and a tumor cell expressing the gene is administered as a vaccine; (9) vaccines wherein a tumor antigenic gene is integrated into a virus or a bacterium for infection of a patient; (10) vaccines wherein a live tumor cell, a tumor antigenic peptide or an extract of a tumor cell is administered, and separately a great amount of a cytokine is administered (Rosenberg, S. A., et al., Nature Med., 4, pp. 321-327, 1998), or wherein a cytokine is formulated into a controlled release preparation and administered (Golumbek, P. T., et al., Cancer Res., 53, pp. 5841-5844, 1993) and the like.
However, any of the above tumor vaccines is advantageous from some aspects while disadvantageous from other points of view. For example, Method (1) can only be applied to tumors which express a specific major histocompatibility complex (hereinafter abbreviated as “MHC”, and for Class I referred to as “MHC-I” and for Class II as “MHC-II”) that meets to an identified tumor antigenic peptide. The human MHC is highly diverse, and consequently, clinical cases are very limited in which those tumor antigenic peptides can meet the MHC. To overcome the problem, Method (2) using a tumor tissue extract containing an unidentified tumor antigenic peptide has been developed. However, only a trace amount of the tumor antigenic peptide can be extracted from tumor tissues, and it is often impossible to concentrate the extract when the amount of the tumor as a raw material is small. Therefore, the extract cannot be administered in a large amount such as identified and synthesized tumor antigen peptides, and effects are limited.
Where a tumor antigenic peptide is bound to an antigen-presenting cell beforehand, such as in Method (3), a high CTL activating effect is obtained. However, peripheral blood or bone marrow for isolation and preparation of the antigen-presenting cell, especially a dendritic cell having strong antigen presenting capability, should be derived from the patient who bears the tumor and is to be applied with the tumor vaccine therapy to prevent dangerous graft-versus-host-disease (hereinafter abbreviated to “GVHD”), which requires a highly skilled technique and is complicated. Methods (4) and (5) have the same problem as that of Method (3), and in addition, a fusion process is very complicated in Method (5). Although there is no concern about the risk of GVHD in Method (6), an efficiency of the introduction of the tumor antigen into the antigen-presenting cell is sometimes not successfully high, and a relatively great amount of the tumor antigen is required to prepare the tumor vaccine.
Method (7) is also complicated and costly because the tumor cells are obtained by mass culture, and moreover, the method has a problem in that the amount of the tumor antigen contained in the tumor cells per se is very small. This method is known to be successful in tumor cells with high antigenicity when treatment with poly(L-lysine) is applied (Naito, M. and Seno, S., Cell Biol. International Rep., 5, pp. 675-681, 1981). However, the method remains unsuccessful in tumor cells with low antigenicity. Genetic therapies of Methods (8) and (9) are extraordinarily complicated in procedures to obtain approval for the treatment, as well as in therapeutic operations. Method (10) is promising at present; however, especially in the method of Rosenberg et al., a huge amount of interleukin-2 simultaneously administered causes a severe side effect, and clinical results for tumors treatment are sometimes not satisfactory. Even when cytokines are formulated as controlled release preparations by the method of Golumbek et al., a complication still remains in preparation of X-ray-irradiated live tumor cells.
The tumor vaccine is desirably provided in a form that can be handled as easily as possible. From this point of view, methods involving administration of live tumor cells or antigen-presenting cells as a part of a tumor vaccine have problems in that they are technically very complicated as operations under a live state are required. The operations are further complicated for a genetic therapy. When tumor antigenic peptides are known, the peptides can be synthesized in large quantities for administration. However, there are a large variety of tumor antigenic peptides, and additionally, due to restriction from MHC molecules of a patient individual, it often cannot be appropriately determine which of tumor antigenic peptides is applicable to the patient individual, which may limit the application. When a tumor antigenic protein is used instead of the tumor antigenic peptide, the protein is processed in the antigen-presenting cells and then a tumor antigenic peptide that meets the MHC is selected. Accordingly, the method is not restricted by the MHC of a patient individual to be treated. However, this method has a problem in that purification and large-scale preparation of the tumor antigenic protein, per se, is difficult.
As a method for inducing CTL, a method is known in which CTL is induced from peripheral blood mononuclear cells on a fixed tumor tissue obtained by removing paraffin from pathological sections (Liu, S. Q. et al., Nature Med., 2, pp. 1283-1283, 1996). Generally, when an antigenic protein in a soluble state is provided to antigen-presenting cells, the protein has a high stimulating effect on liquid immunity that links to production of antibodies by binding antigenic proteins-derived antigen peptides to MHC-II, whereas the protein has a low stimulating effect on cellular immunity that activates killer cells by binding antigenic proteins-derived antigen peptides to MHC-I. Falo et al. conducted induction of CTL that react to ovalbumin-derived antigenic peptides by binding ovalbumin as a foreign protein with strong antigenicity to iron powder and administering the product to mice without addition of an adjuvant (Falo, Jr., L. D., et al., Nat. Med., 1, pp. 649-653, 1995).
The inventors of the present invention found that CTL can be induced efficiently from peripheral blood lymphocytes of the same individual by fixing soluble tumor antigenic proteins on fine polystyrene beads and subjected the product as fine solids to phagocytosis by antigen-presenting cells in human peripheral blood mononuclear cells in a cell culture system in vitro (Kim, C., et al., Cancer Immunol. Immunother., 47, pp. 90-96, 1998). It is also known that dead cell-derived antigens can efficiently induce immune responses thousands folds stronger when the antigens are phagocytosed in the state of dead cells by immature dendritic cells than when the antigens are not phagocytosed (Inaba, et al., Lecture SI-3-3, Japanese Immunology Society, Dec. 2, 1998).